DESCRIPTION: (Applicant's Abstract) Cannabinoid receptors are molecular targets for marijuana, the most widespread illegal drug of abuse in Western societies. Cannabinoid receptors are densely expressed in areas of the central nervous system (CNS) that participate in the control of pain perception, appetite, memory, cognition and movement. Such functions are strongly affected by cannabinoid drugs, with consequences that include analgesia, appetite stimulation, euphoria and memory impairment. Although the pharmacology of cannabinoid drugs is now beginning to be understood, the endogenous signaling system by which cannabinoid receptors are physiologically engaged remains largely unexplored. An endogenous ligand for cannabinoid receptors, anandamide, has been described. Anandamide is released from neurons on depolarization, and it undergoes a rapid process of biological inactivation. The molecular mechanisms underlying such inactivation are, however, still only partially understood. On the basis of our preliminary findings, we propose to test the hypothesis that carrier-mediated transport into neural cells constitutes a prominent route of anandamide inactivation in the CNS. To test this hypothesis it will be necessary to show that neural cells accumulate anandamide by a mechanism that fulfills the criteria of a carrier-mediated transport (i.e., time- and temperature-dependence, high affinity, substrate selectivity, and inhibition by specific agents). Moreover, it will be necessary to demonstrate that anandamide transport participates in terminating anandamide responses at its sites of action in the CNS. The first aim of the proposed research is to investigate the mechanisms responsible for anandamide transport. Preliminary experiments suggested that rat brain neurons and astrocytes in primary culture accumulate anandamide by a carrier-mediated transport system. The proposed studies will characterize the mechanism of anandamide transport in these cells. The second aim of our proposed research is to determine what are the structural determinants for anandamide transport. Preliminary experiments have shown that anandamide transport is highly specific. We will explore the structure activity relationship of anandamide transport, by designing and synthesizing novel anandamide-like compounds and test them for their ability to act as transport substrates or inhibitors. The third aim of our proposal is to define the properties, topographic distribution and functional roles of anandamide transport in CNS. Initial experiments have indicated that rat brain slices accumulate anandamide by a transport mechanism similar to the one we have identified in cultured cells. The proposed research will characterize this transport, determine its topographic distribution in CNS, and investigate its role in the elimination of endogenous anandamide released by physiologically relevant stimuli. These studies will set the stage for the molecular characterization of the anandamide transporter protein and for the development of potent and selective anandamide transport inhibitors. In conclusion, by demonstrating that transmembrane transport participates in anandamide inactivation, our studies will shed new light on the mechanisms of marijuana abuse and help develop novel strategies in the therapy of neurological, psychiatric and substance abuse disorders.